Injectable Osteogenic Formula and Method of Using Same

ABSTRACT

Formulations and methods for growing bone in a site specific location using an osteogenic molecule such as a prostaglandin, and a delivery vehicle which is preferably a polymer matrix.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority of Provisional application Ser. No.60/693,391 filed on Jun. 23, 2006, the disclosure of which isincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to growing bone in a site-specific location.

BACKGROUND OF THE INVENTION

The ability to grow bone at a site-specific location, in a minimallyinvasive manner, would have a profound impact on the health and qualityof life for up to 50 million Americans who suffer bone defects anddiseases. These diseases include post craniotomy resorption, periodontaldisease, degenerative disk disease, osteoporosis, and asepticosteolysis. Together, these diseases cost the United States HealthcareSystem in excess of $124 billion annually.

Cranial

Over 750,000 craniotomies are performed every year in the United States,to treat a variety of disorders including tumors, traumas, vascularlesions, seizures, decompression, cranioplasty, infections, intracranialcysts, and nerve decompression. Despite this, the skull has been one ofthe most difficult regions in which to use autograft techniques becauseof the cranium's propensity for resorption. Many materials and methodshave been used including autologous bone grafts, metal plates (titanium,tantalum, stainless steel), hydroxyapatite cement, andmethylmethaerylate, each with significant drawbacks.

Periodontal Disease.

The most common treatment for periodontal disease and tooth loosening isextraction and dental implantation or dentures. Over 20% of Americans,approximately 56 million people, have periodontal disease. Periodontaldisease accounted for 10% of all dental costs in 1985.

Spinal Disorders

Spine disorder treatments are slowly trending toward minimally invasivetechniques for penetrating past the muscle tissues surrounding thespine. Despite this, the solutions to degenerative disks (fusion,herniated disk repair and disk replacement) are still highly invasiveprocedures resulting in extensive surgical trauma and prolonged recoverytimes. Over 1 million spinal surgeries were performed last year and thisnumber continues to rise.

Joint Replacements

Over ½ million knee and hip replacement surgeries are performed in theUnited States every year. The typical age of these patients is 65 yearsdespite the fact that the average age of pain symptom onset is 40 years.The reason for this disparity is that the implants cause bone resorptionat the interface, making their maximum useful life less than 15 years.

Overall, the ability to induce rapid, localized bone growth would have asubstantial beneficial effect in all the above conditions by reducingmorbidity, hospitalization time, recovery time, and costs. The abilityto grow bone at specific sites would also have a substantial positiveimpact on the treatment of bone fractures resulting from osteoporosis.It is estimated that over 24 million people suffer from osteoporosis,resulting in 3 million fractures per year in the United States. In 1995osteoporotic fractures were estimated at $13.8 billion in direct medicalexpenses. Disorder Annual U.S. Cases Craniotomies 750,000 PeriodontalDisease 28,000,000 Spinal Surgeries 1,000,000 Aseptic Osteolysis 500,000Osteoporosis 3,000,000 Total 33,250,000

Bone Morphogenic Proteins (BMPs) are osteogenic compounds. However, BMPscan cause ectopic ossification, making them somewhat difficult and riskyto use.

SUMMARY OF THE INVENTION

The invention comprises formulations and methodologies for treatingdegenerative bone conditions, bone fractures, and other bone-relatedconditions by combining an osteogenic compound with a delivery vehicle.The resulting combination may be injected or otherwise applied to (suchas by an implant or other device, or by applying it to a site duringsurgery) a specific site, and cause bone growth at that site.

This invention features an osteogenic formulation comprising anosteogenic compound and a delivery vehicle. The osteogenic compound ispreferably prostaglandin (PGE), and may comprise PGE1 and/or PGE2. Onereason that prostaglandins are the preferred osteogenic compounds forthe invention is that they do not induce bone synthesis at ectopic sitesby non-bone cells.

The delivery vehicle may comprise a biodegradable matrix, whichpreferably comprises one or more polymers. Two preferred polymers arehyaluronic acid or a salt thereof, and/or a poly-glutamic acid.

The invention also features a method of employing an osteogeniccompound, comprising providing prostaglandin (PGE) osteogenic compound,providing a biodegradable polymer matrix delivery vehicle for the PGE,mixing the PGE and the delivery vehicle, and delivering the mixture to asite. The PGE may comprise PGE1 and/or PGE2. The polymer preferablycomprises hyaluronic acid or a salt thereof, and/or a poly-glutamicacid. The delivery is preferably accomplished with a syringe, but asdescribed above can be accomplished by other know means forsite-specific delivery of a treatment vehicle.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following is a description of the preferred embodiments of theinvention. The half-life of prostaglandin E1 (PGE1) is measured inminutes. PGE1 is rapidly metabolized in the lungs, therefore thedelivery method is critical to widespread commercial use of PGE1 as anosteogenic compound. The invention is designed to stabilize and holdPGE1, which is by far the most potent osteogenic agent discovered todate, and deliver it via a gelatinous polymer matrix of hyaluronic acid,a material that is itself modestly osteogenic. PGE2 is less osteogenicthan PGE1 but may still be useful in the invention. The matrix allowsthe osteogenic compound (preferably a prostaglandin) to make physicalcontact with the targeted bone surface, thus stimulating only the cellsrequired for the healing process. The matrix releases the compound overa period of time so that the stimulation of the cells is maintained overa period of time until the process of osteogenesis is completed. Boththe matrix and the compound are reabsorbed by the body. Because thematerial may be delivered by a syringe, more than one treatment can bereadily accomplished. No surgical procedures are required.

The delivery matrix is a biodegradable polymer The polymer should begelatinous in nature and water soluble. This includes polymers such ascarboxymethylcellulose, poly-glutamic acid (PGA), but preferably thepolymer is hyaluronic acid. Hyaluronic acid is biocompatible and isitself mildly osteogenic.

The following are examples of producing the various preferredformulations.

Example of Preferred Formulation

PGE1 is dissolved in pure water. In the event that the PGE1 won'tcompletely dissolve, pure ethanol may be added until all the PGE1 isdissolved. The concentration of PGE1 may be varied as necessary.

Hyaluronic acid is hydrated in pure water. Hyaluronic acidconcentrations may also be varied, but a concentration of greater than30 mg/ml in pure water works well and may thus be diluted with the PGE1solution.

The PGE solution is combined with the hyaluronic acid gel and allowed tomix. Mixing may be achieved in a mixer, a beaker with a stir bar andmagnetic stirrer, or even by coupling two syringes together, eachcontaining one of the two solutions. Solutions should be mixed untilhomogeneous. In addition, salts of hyaluronic acid may be used to varythe release of the PGE1. Sodium hyaluronate, calcium hyaluronate, andeven ferric hyaluronate may be used. As the valence of the countercation increases (i.e., Na+, Ca++, Fe+++and so forth) the half life ofhyaluronate increases, and the release rate of PGE1 decreases.

Following are examples of purified hyaluronic acid andpoly-gamma-glutamic acid that may be produced for use as the matrix forthe invention.

Example of Hyaluronic Acid Purification

1. Rooster combs are sliced and placed in ethanol. The ethanol ischanged daily until it is no longer cloudy. Three days, 6 liters ethanolused.

2. The ethanol is drained and the combs are placed in water with anantiseptic (Thymol) to prevent microbial growth.

3. The combs are mixed at less than 100° C. overnight or until thesolution viscosity exceeds 500 cps. Steps 2 and 3 together take 3 daysand use no ethanol.

4. The combs are strained from the extract. The extract is treated withNaCl to a final concentration of 0.2M.

5. The extract is centrifuged and added to 3 volumes of ethanol and theresulting stringy white precipitate is removed and stored under ethanol.Steps 4 and 5 together take I day and use 3 liters ethanol.

6. Dissolve precipitate in DI water to approximately 1.5 mg/mlconcentration. Though the actual concentration will change later, 0.75to 5.0 mg/ml HA may be successfully precipitated with ethanol (andNaCl).

7. Add 100 ml of chloroform to every 1 liter of solution, mix overnightand centrifuge for 5 minutes at 4,000 RPMs. This step removes residualfats, lipids, certain proteins and other materials that have been foundto inhibit the Pronase® step. Steps 6 and 7 together take 2 days and useno ethanol.

8. Add the aqueous portion to a temperature-controlled reactor, add anantiseptic (Thymol), <0.5mM CaCl₂, heat to 370° C., adjust pH to 8.0 andadd Pronase®. These are optimum Pronase® conditions per CalBiochem,Pronase® manufacturers.

9. Maintain pH at 8.0 via pH control and the addition of 0.2M Trisbuffer. Run until no more Tris is required (typically overnight). Thishydrolyzes proteins not removed by chloroform, as well as the linkproteins responsible for binding HA to other GAG's. Steps 8 and 9together take 1 day and use no ethanol.

10. Make up a solution of 100 mls of 2% CPC and 0.3M NaCl. Adjust thereactor contents to 0.3M, and add the CPC/NaCl solution to the reactor.It will change color from opaque to yellow. Allow it to mix for 15minutes. Filter the reactor contents through a membrane filter (0.2micron PES filter) and collect in a flask.

This causes DNA, chondriotin sulfate, heparin and other non-HA GAG's tocomplex and precipitate. They are subsequently removed by filtration.This step takes one-half day, and no ethanol.

11. Using a Pall-Filtron 30kDa MWCO PES membrane, diafilter the solutionagainst 5 volumes of 0.3M NaCl. This removes amino acids, peptides,Pronase®, CPC and other low MW contaminants.

12. Either precipitate with ethanol and dry under vacuum, or lyophilizethe contents of the flask. This is the best way to store material untilformulation. Formulation strength cannot be achieved through TFD at thistime. Steps 11 and 12 together take one-half day and use 3 litersethanol.

13. Formulate to 10 mg/ml and verify properties against the traditionalprocess. 10 mg/ml is a simple HA concentration that has been used oftenin the industry. One day, no ethanol. The following describes theequipment used for diafiltration: DF Parameter Value 1. Three membranesetup (Used to determine MW cutoff for diafiltration) Membrane TypeOmega Polyethersulfon Channel Depth 40 mil Membrane Area 0.045ft²/channel Number of Channels 3 in parallel Trans-Membrane Pressure(TMP) 18.5 psig Pressure drop across membrane 2.5 psig Cross Flow Rate200 ml/min/channel 2. Single membrane DF experimental setup (Used topurify HA from rooster comb) Membrane Type Omega PolyethersulfonMembrane Area 1.0 ft² Trans-Membrane Pressure (TMP) 8.0 psig Cross FlowRate 1,000 ml/min MWCO 30k Type Centramate Configuration Open Channel

The pump used to performed the diafiltration was a Cole-ParmerMasterflex® L/S® Precision Standard Tubing Pump capable of over 1700ml/min, SKU# EW-77911-00.

Example 1 PGA Using Preferred Fermentation Method, and Purification toMedical Grade

Bacillus licheniformis ATCC 9945a was grown in Medium E. Thefermentation was carried out at small scale, in shake flasks, at 37° C.Aeration was provided by diffusion. When the viscosity stopped rising(typically after about 3-5 days of fermentation), the fermentation brothwas buffer exchanged via diafiltration using a filter with a molecularweight cut off (MWCO) of 30 kDa. The mixture of cells and PGA was thenbuffered in citric acid, and micro-filtered using a filter with anopening of 0.22 microns, to remove the host cells.

The filtrate was neutralized, and buffer exchanged with pure water aridconcentrated via diafiltration using a filter with a MWCO of 30 kDa.Material from this purification may be sterile filtered.

To describe the process in more detail, when the viscosity stoppedrising, the fermentation broth was re-circulated through an OmegaPolyethersulfon ultra-filtration cartridge by Pall Corporation with a0.2 micron pore size. Once collected, the filtrate was re-circulatedusing an Omega Polyethersulfon ultra-filtration cartridge by PallCorporation with a 0.16 micron pore size. The filtrate was collected andre-circulated through an Omega Polyethersulfon ultra-filtrationcartridge by Pall Corporation with a 30kda MWCO pore size. Fivediafiltration volumes of solution were processed. At the end, theretentate was collected, sterilized by passing through a 0.22 micronfilter, and precipitated in sterile ethanol and stored.

Material from this example has been used in rats in subsequentexperiments with no inflammatory response. The molecular weight wasdetermined to be 2 million Daltons using the following analytical MALLSmethod described in the Stock thesis that is incorporated by referenceherein. PGA was dissolved at a concentration of 1 mg/ml in 0.1 M citricacid, pH 2 to 3, with 0.05% sodium azide. The sample was degassed and0.2 milliliters was injected at a flow rate of 0.5 mls/min. The SEC canutilize a TossoHaas TSK G5000PWXL, G6000PWXL, Waters Ultrahydrogel 1000or 250. A Dawn DSP laser photometer from Wyatt technologies inconjunction with a Waters differential refractometer is used fordetection,

This process is capable of making high molecular weight (when measuredas described) poly-gamma-glutamic acid at purities up to and includingpharmaceutical grade.

Example 2 PGA From Another Commercial Source Purified

A sample reported to be poly-gamma-glutamic acid in excess of 1 millionDaltons was received from an offshore commercial supplier. The viscosityof a sample of known concentration seemed to be lower than would be thecase if the PGA was indeed of the reported molecular weight. Analysiswas impossible due to the large amount of contaminants, as evidenced bythe off-white color noted when the sample was hydrated, and the factthat the hydrated sample had an odor similar to fermentation broth.

This material was re-circulated through an Omega Polyethersulfonultra-filtration cartridge by Pall Corporation with a 0.2 micron poresize. Once collected, the filtrate was re-circulated using an OmegaPolyethersulfon ultra-filtration cartridge by Pall Corporation with a0.16 micron pore size. The filtrate was collected and re-circulatedthrough an Omega Polyethersulfon ultra-filtration cartridge by PallCorporation with a 30kda MWCO pore size. Five diafiltration volumes ofsolution were processed. The resulting material was clear and odorless,supporting the production of low molecular weight, high purity PGA.

Example 3 PGA

Bacillus licheniformis ATCC 9945a was grown in Medium E. Thefermentation was carried out at small scale, in shake flasks, at 37° C.Aeration was provided by diffusion. When the viscosity stopped rising,the fermentation broth was buffer exchanged via diafiltration using afilter with a molecular weight cut off (MWCO) of 30 kDa. The mixture ofcells and PGA was then buffered in citric acid, and micro-filtered usinga filter with an opening of 0.16 microns.

The filtrate was neutralized, and buffer exchanged with pure water andconcentrated via diafiltration using a filter with a MWCO of 30 kDa.Material from this purification may be sterile filtered. Material fromthis example has been used in rats in subsequent experiments with noinflammatory response. The molecular weight was determined to be 2million Daltons using the method described above in conjunction withexample 1.

Example 4 PGA

Bacillus licheniformis ATCC 9945a was grown in Medium E. Thefermentation was carried out at small scale, in shake flasks, at 37° C.Aeration was provided by diffusion. When the viscosity stopped rising,the pH of the fermentation broth was lowered to 2 by the addition ofHCl. The cells were then removed by passing the broth through a 0.22micron TFF filter and collecting the filtrate. The filtrate was thenneutralized, and buffer exchanged with pure water and concentrated viadiafiltration using a filter with a MWCO of 30 kDa. Material from thispurification may be sterile filtered. Material from this example hasbeen used in rats in subsequent experiments with no inflammatoryresponse. The molecular weight was determined to be 2 million Daltonsusing the method described above in conjunction with example 1.

Example 5 PGA

Bacillus licheniformis ATCC 9945a was grown in Medium E. Thefermentation was carried out at small scale, in shake flasks, at 37° C.Aeration was provided by diffusion. When the viscosity stopped rising,the pH of the fermentation broth was lowered to 2 by the addition ofHCl. The cells were then removed by centrifugation at a speed over10,000×g. The supernatant was then neutralized, and buffer exchangedwith pure water and concentrated via diafiltration using a filter with aMWCO of 30 kDa. Material from this purification may be sterile filtered.Material from this example has been used in rats in subsequentexperiments with no inflammatory response. The molecular weight wasdetermined to be 2 million Daltons using the method described above inconjunction with example 1.

1. An osteogenic formulation, comprising: an osteogenic compound; and adelivery vehicle.
 2. The osteogenic formulation of claim 1, wherein thecompound comprises prostaglandin (PGE).
 3. The osteogenic formulation ofclaim 2 in which the compound is PGE1.
 4. The osteogenic formulation ofclaim 2 in which the compound is PGE2.
 5. The osteogenic formulation ofclaim 1, wherein the delivery vehicle comprises a biodegradable matrix.6. The osteogenic formulation of claim 5, wherein the biodegradablematrix comprises a polymer.
 7. The osteogenic formulation of claim 6,wherein the polymer comprises hyaluronic acid or a salt thereof.
 8. Theosteogenic formulation of claim 6, wherein the polymer comprises apoly-glutamic acid.
 9. An osteogenic formulation, comprising:prostaglandin (PGE) osteogenic compound; and a biodegradable polymermatrix delivery vehicle for the PGE.
 10. The osteogenic formulation ofclaim 9, wherein the polymer comprises hyaluronic acid or a saltthereof.
 11. The osteogenic formulation of claim 9, wherein the polymercomprises a poly-glutamic acid.
 12. A method of employing an osteogeniccompound, comprising: providing prostaglandin (PGE) osteogenic compound;providing a biodegradable polymer matrix delivery vehicle for the PGE;mixing the PGE and the delivery vehicle; and delivering the mixture to asite.
 13. The method of claim 12 in which the PGE comprises PGE1. 14.The method of claim 12 in which the PGE comprises PGE2.
 15. The methodof claim 12 in which the polymer comprises hyaluronic acid or a saltthereof.
 16. The method of claim 12 in which the polymer comprises apoly-glutamic acid.
 17. The method of claim 12 in which delivery isaccomplished with a syringe.
 18. A method of employing an osteogeniccompound, comprising: providing prostaglandin (PGE) osteogenic compound;providing a biodegradable polymer matrix delivery vehicle for the PGEcomprising hyaluronic acid or a salt thereof; mixing the PGE and thedelivery vehicle; and delivering the mixture to a site with a syringe.